Cholera is an ancient disease in the midst of a modern resurgence, with cases reported to WHO by 65 member nations in Asia, Africa, the Americas, and Europe in 1997. With the appearance of V. cholerae 0139 Bengal and the recent identification of epidemic-associated 037 strains, there is increasing recognition that modifications in bacterial surface polysaccharides play a key role in the emergence of new, epidemic-associated strains. While the number of constituent sugars appears to be relatively small (less than 25), there are hundreds of O antigen/capsule types recognized among V. cholerae and other Vibrionaceae strains. Initial phylogenetic studies suggest that serotype and/or capsule type are independent of phylogeny. We hypothesize that there is frequent horizontal/sexual transfer and rearrangement of surface polysaccharide biosynthetic gene cassettes in V. cholerae, providing an opportunity for periodic emergence of new, pandemic cholera strains. Making use of our collection of Vibrionaceae (including the Smith Vibrio Reference Laboratory/serotype collection) and ongoing strain acquisition, we propose: 1) To describe and define the genetic relatedness of a 192-strain V. cholerae test collection, utilizing sequence analysis of conserved genetic elements (multilocus sequence typing) including genes used in multilocus enzyme electrophoresis/zymovar analysis. 2) To identify and characterize V. cholerae O antigen and capsular polysaccharides for stains within this collection, as a basis for assessing horizontal transfer of surface polysaccharide genes. This will include a) determination of sugar composition of O-antigen side chain and capsule; and b) identification and correlation of sugars with specific biosynthetic genes/gene combinations. 3) To assess the extent of genetic variability in surface polysaccharides plausibly available to V. cholerae, and, in particular, to V. cholerae strains with pandemic potential. For these latter studies we will a) correlate genetic relatedness (as determined above) with transfer/acceptance of specific polysaccharide genes; b) determine the placement and organization of biosynthesis genes for the sugars of the O side chain and capsule; and c) assess the nucleotide sequence divergence of the most commonly identified polysaccharide biosynthesis genes. Taken together, these studies will substantively expand our understanding of the evolutionary biology of V. cholerae, giving us a much better appreciation of the potential for changes in V. cholerae surface polysaccharides and, in turn, for the emergence of V. cholerae strains which can form the basis for new cholera pandemics.